Interest in the substrate specificity of bacterial dioxygenases stems from initial studies on the degradation of benzene and toluene more than 25 years ago. A mutant strain of Pseudomonas putida (strain F39/D) was shown to oxidize benzene and toluene to cis-1,2-dihydroxycyclohexa-3,5-diene (cis-benzene dihydrodiol) and cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol), respectively (D. T. Gibson, et al., Biochemistry, 1970, 9, 1631-1635; D. T. Gibson, et al., Biochemistry, 1970, 9, 1626-1630; and V. M. Kobal et al., J. Am. Chem. Soc., 1973, 95, 4420-4421).
The enzyme catalyzing these reactions, toluene dioxygenase (TDO), is capable of producing enantiomerically pure cyclohexadiene cis-diols from a wide range of aromatic substrates. D. T. Gibson, et al., Microbial Degradation of Organic Compounds (Gibson, D. T., ed.), pp. 181-251, Marcel Dekker, New York, N.Y. (1984); D. T. Gibson, et al., Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology, (Silver, S. et al., ed.), pp. 121-132, American Society for Microbiology, Washington D.C. (1990); G. N. Sheldrake, Chirality in Industry: the Commercial Manufacture and Application of Optically Active Compounds (Collins, A. N. et al., eds.), pp. 127-166, John Wiley and Sons, Chichester, UK (1992); Stabile, M. R., Ph.D. thesis. Virginia Polytechnic Institute and State University, Blacksburg, Va. (1995); and D. R. Boyde and G. N. Sheldrake, Nat. Prod. Rep. 1988, 15, 309-324.
In contrast to the body of work relating to TDO, relatively little attention has been paid to the related enzyme naphthalene dioxygenase. Cells of Pseudomonas sp NCIB 9816-4 contain an inducible multi-component enzyme system designated NDO, which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (D. M. Jerina et al. Arch. Biochem. Biophys. 1971, 142, 394-396). NDO has a relaxed substrate specificity and catalyzes the deoxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respective cis-diols.
The potential of NDO to form products of opposite chirality to those formed by TDO was first noted in 1988 during studies on the oxidation of indan. The major product formed by TDO was (xe2x88x92)-(1R)-indanol (84% enantiomeric excess [ee]) whereas NDO produced (+)-(1S)-indanol ( greater than 92% ee) (L. P. Wackett et al., Biochemistry, 1988, 27, 1360-1367. Subsequent studies with NDO revealed further differences in substrate specificity and suggested that this enzyme is an additional source of chiral intermediates and synthons for the enantiospecific synthesis of biologically active products. S. M. Resnick et al. Journal of Industrial Microbiology, 1996, 17, 438-457.
NDO belongs to a family of bacterial enzymes that have an essential role in the recycling of carbon in nature. These enzymes are especially important in the degradation of aromatic hydrocarbons and related environmental pollutants. Knowledge of the NDO reaction mechanism is thus important in the development of bioremediation strategies for cleaning up environments contaminated with hazardous aromatic compounds. An attractive alternative to bioremediation is the application of xe2x80x98green chemistry,xe2x80x99 which refers to the production of industrial chemicals by processes that do not generate hazardous waste. For example, a recombinant strain of Escherichia coli expressing NDO, has been used to synthesize indigo dye from glucose. cis-Arene diols produced by NDO and toluene dioxygenase have been used in the synthesis of many products of biological and economic importance.
Knowledge of the types of reactions catalyzed by NDO and the range of substrates oxidized by NDO is based largely on biotransformation studies with cis-naphthalene dihydrodiol dehydrogenase (DDH) mutants, recombinant strains expressing NDO and purified NDO components. Pseudomonas sp 9816/11 is a DDH mutant of strain 9816-4 (G. M. Klecka and G. T. Gibson, Biochem J., 1979, 180, 639-645) which accumulates cis-naphthalene-1,2-dihydrodiol when induced cells are incubated with naphthalene and a suitable carbon source (D. S. Torok, J. Bacteriol. 1995, 177, 5799-5805. Studies with purified dioxygenase components have been crucial in the identification of reactions catalyzed by NDO in the absence of other host-associated enzyme activities which, through subsequent catalysis, have the potential to affect product distribution and/or stereochemistry.
In addition to cis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation), O- and N-dealkylation and sulfoxidation reactions. S. M. Resnick et al. Journal of Industrial Microbiology, 1996, 17, 438-457. Many of the reactions catalyzed by NDO and other microbial dioxygenases yield hydroxylated compounds that can serve as chiral intermediates or chiral synthons for a variety of compounds of interest to pharmaceutical and specialty chemical industries.
Despite the wide range of useful oxygenated materials that can be prepared with TDO and NDO, there is currently a need for additional oxygenated chiral synthons that can be used to prepare therapeutically useful compounds, or useful intermediates. In particular, there is a need for additional chiral synthons that differ from the TDO and NDO products by absolute configuration or by the site of oxygenation. There is also a need for new methods to prepare hydroxylated aryl compounds for use in the polymer, resin, pharmaceutical or rubber industry, which generate less industrial waste than currently available methods. Further, there is a need for novel enzymes possessing structures, stabilities, or reactivities that differ from the native enzymes.
The crystal structure of NDO has recently been published by B. Kauppi et al. Structure, 1998, 6, no. 5, 571-586. Based on this structure, the amino acid at position 352 is located at the active site of NDO. As described hereinbelow, site-directed mutagenesis was used to construct DNA molecules that encode NDO mutants having amino acid substitutions at position 352. Changing the amino acid at position 352 from phenylalanine to valine provided an enzyme (SEQ ID NO:2, encoded by SEQ ID NO:1) that gives a change in the preferred absolute configuration of the 1,2-dihydroxy-1,2-dihydronaphthalene formed from naphthalene. This enzyme also gave a change in the regioselectivity of the products obtained from oxidation of biphenyl and phenanthrene.
Accordingly, the invention provides an NDO or NDO related complex comprising a plurality of polypeptides, wherein the complex comprises at least one alpha-subunit polypeptide that comprises: 1) a substituted amino acid (e.g. valine or leucine) at the position corresponding to position 352 in NDO, 2) a substituted amino acid at the position corresponding to position 201, 202, 260, 316, 351, 358, 362, or 366 in NDO, or 3) a substituted amino acid at the position corresponding to position 352 in NDO, and a substituted amino acid at the position corresponding to position 201, 202, 260, 316, 351, 358, 362, or 366 in NDO; or a catalytically active fragment thereof. The complexs of the invention can preferably be isolated and purified.
The invention also provides an isolated and purified polypeptide having Swiss-prot data base Accession Number P23094 that comprises an amino acid other than phenylalanine at position 352, or a catalytically active variant, or a catalytically active fragment thereof. Preferably, the amino acid at position 352 is a naturally occurring amino acid. More preferably, the polypeptide is SEQ ID NO:2, 32, 33, 34, 35, or 36.
The invention also provides an isolated and purified NDO related polypeptide wherein the amino acid at the position corresponding to position 352 in NDO has been replaced with another amino acid, or a catalytically active fragment or catalytically active variant thereof. Preferably, the amino acid at the position corresponding to position 352 in NDO is a naturally occurring amino acid. More preferably, the amino acid at the position corresponding to position 352 in NDO is valine.
Site-directed mutagenesis was also used to construct DNA molecules that encode NDO mutants having an amino acid substitution at position 201, 202, 260, 316,351, 352, 358, 362, or 366. Accordingly, the invention provides an isolated and purified NDO wherein the amino acid at position 201, 202, 260, 316, 351, 352, 358, 362, or 366 has been replaced with another amino acid, or a catalytically active variant, or a catalytically active fragment thereof.
Changing the amino acid at position 352 in NDO from phenylalanine to valine provided an enzyme (SEQ ID NO:2, encoded by SEQ ID NO:1) that gives a change in the preferred absolute configuration of the 1,2-dihydroxy-1,2-dihydronaphthalene formed from naphthalene. This enzyme also gave a change in the regioselectivity of the products obtained from oxidation of biphenyl and phenanthrene The invention also provides an isolated and purified DNA segment encoding a polypeptide of the invention, or a variant or fragment thereof.
The invention also provides a primer or probe having about 80% nucleic acid sequence identity with a DNA segment encoding a polypeptide of the invention, or a variant or fragment thereof.
The invention also provides an expression cassette comprising a promotor operably linked to a DNA segment encoding a polypeptide of the invention or a variant or fragment thereof.
The invention also provides a host cell, the genome of which is augmented by a DNA segment encoding a polypeptide of the invention, or a catalytically active variant or fragment thereof.
The invention also provides a method to produce a catalytically active polypeptide comprising culturing a host cell transformed with a DNA segment encoding a polypeptide of the invention, or a catalytically active variant or fragment thereof, so that the host cell expresses the polypeptide, variant or fragment.
The invention also provides a method for preparing cis-naphthalene dihydrodiol (e.g. (xe2x88x92)-(1S,2R)-cis-naphthalene dihydrodiol) comprising contacting naphthalene with a polypeptide of the invention, or a catalytically active variant or fragment thereof.
The invention also provides a method for preparing cis-naphthalene dihydrodiol (e.g. (xe2x88x92)-(1S,2R)-cis-naphthalene dihydrodiol) comprising contacting a host cell of the invention with naphthalene.
The invention also provides a method for preparing cis-biphenyl-3,4-dihydrodiol (e.g. (xe2x88x92) or (+) cis-biphenyl-3,4-dihydrodiol) comprising contacting biphenyl with a polypeptide of the invention, or a catalytically active variant or fragment thereof. The method may optionally further comprise dehydrating the cis-biphenyl-3,4-dihydrodiol to give 4-hydroxybiphenyl.
The invention also provides a method for preparing cis-biphenyl-3,4-dihydrodiol (e.g. (xe2x88x92)-cis-biphenyl-3,4-dihydrodiol) comprising contacting a host cell of the invention with biphenyl. The method may optionally further comprise dehydrating the (xe2x88x92)-cis-biphenyl-3,4-dihydrodiol to give 4-hydroxybiphenyl.
The invention also provides a method for preparing cis-phenanthrene-1,2-dihydrodiol (e.g. cis-(1S,2R)-phenanthrene-1,2-dihydrodiol) comprising contacting phenanthrene with a polypeptide of the invention, or a catalytically active variant or fragment thereof.
The invention also provides a method for preparing cis-phenanthrene-1,2-dihydrodiol (e.g. cis-(1S,2R)-phenanthrene-1,2-dihydrodiol) comprising contacting a host cell of the invention with phenanthrene.
The invention also provides a method to oxidize an aromatic compound to a corresponding dihydrodihydroxy compound comprising contacting the aromatic compound with a polypeptide of the invention, or a catalytically active variant or fragment thereof.
The invention also provides a method to oxidize an aromatic compound to a corresponding dihydrodihydroxy compound comprising contacting the aromatic compound with a host cell of the invention.
The invention also provides a method to prepare an optically active cis-cyclohexadiene of formula (I): 
wherein one of R1 and R2 is hydrogen and the other is phenyl, 2-phenylvinyl, 2-phenylethynyl, or vinyl, wherein any phenyl ring may optionally be substituted with 1, 2, or 3 substituents independently selected from the group consisting of hydroxy, halo, carboxy, cyano, nitro, trifluoromethyl, amino, (C1-C6)alkyl, (C1-C6)alkoxycarbonyl, and (C1-C6)alkoxy, comprising contacting a corresponding compound of formula (II): 
with a polypeptide of the invention, or a catalytically active variant or fragment thereof, or with a host cell of the invention. Preferably, R1 is phenyl or 4-hydroxyphenyl. The method may optionally comprise dehydrating the resulting compound of formula I.
The invention also provides a method to prepare cis-1,2-dihydroxyindan comprising contacting indene with a polypeptide of the invention, or a catalytically active variant or fragment thereof, or with a host cell of the invention.
The invention also provides a method to prepare 1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene comprising contacting 1,2-dihydronaphthalene with a polypeptide of the invention, or a catalytically active variant or fragment thereof, or with a host cell of the invention.
The invention also provides a method to prepare 1,2-dihydroxy-1,2-dihydrophenanthrene or 3,4-dihydroxy-3,4-dihydrophenanthrene comprising contacting phenanthrene with a polypeptide of the invention, or a catalytically active variant or fragment thereof, or with a host cell of the invention.
The invention also provides novel compounds and intermediates disclosed herein, as well as crystallized forms of the polypeptides disclosed herein. Preferably, the invention provides a crystallized form of SEQ ID NO:26.
Polypeptides of the invention provide a biosynthetic route to the either enantiomer of cis-naphthalene dihydrodiol, to either enantiomer of cis-biphenyl-3,4-dihydrodiol, and to either enantiomer of cis-phenanthrene-1,2-dihydrodiol. These products can be used in the synthesis of new polymers and pharmaceutical products. For example, arene cis-diols are useful starting materials for stereospecific organic synthesis (S. M. Brown, et al., Organic Synthesis: Theory and Applications (Hudlicky, T., ed.), pp. 113-176, JAI Press, Greenwich, Conn. (1993); and T. Hudlicky and J. W. Reed, Adv. Asymm. Synth. 1995, 1, 271-312). They can undergo a variety of reactions including asymmetric Diels-Alder reactions, epoxidation, photochemical oxygenation, metallation, diol cleavage, diene cleavage, carbene additions and ozonolysis. They have been used to prepare a variety of synthetic products that are not readily obtainable by conventional chemical synthesis. Examples include conduritols, inositol phosphates, pinitol enantiomers, prostanoid and terpene synthons, and complex natural products such as (xe2x88x92)-zeylena and (+)-lycoricidine.